KR20120135366A - Genes enhancing resistance to stress and uses thereof - Google Patents

Abstract

PURPOSE: A composition containing SaDhn gene is provided to enhance resistance to osmotic stress or freezing stress and to improve plant productivity. CONSTITUTION: A Suaeda asparagoides-derived dehydrin SaDhn Suaeda asparagoides dehydrin protein has an amino acid of sequence number 2. A gene encoding SaDhn protein has a base sequence of sequence number 1. A composition for enhancing resistance to osmotic stress and freezing stress in a plant contains the SaDhn gene. A method for enhancing resistance to osmotic stress or freezing stress comprises a step of transforming a recombinant vector containing SaDhn gene into a plant and a step of expressing the SaDhn gene.

Description

Genes that enhance stress resistance and uses thereof

The present invention relates to genes and their use to enhance resistance to osmotic stress and freezing stress.

More specifically, the present invention provides a SaDhn protein similar to dehydrin derived from Suaeda asparagoides , a gene encoding the protein, a recombinant vector comprising the gene, an osmotic stress or freezing including the gene. It relates to a composition for enhancing stress resistance.

Environmental stresses inhibit plant growth and limit yield productivity in many important agricultural sectors, including water stress, low temperature stress, and salt stress. In particular, osmotic stress caused by various external conditions such as dehydration, high salt and cold is the most severe stress for plants.

Since only a slight increase in the resistance to osmotic stress can have a significant effect on agricultural productivity and yield, much research has been conducted on the mechanism of regulation of osmotic stress and related regulatory factors. Recent studies have revealed that plants have precise mechanisms as part of their adaptive response to osmotic stress, and one of the important mechanisms of these adaptive responses is the transcriptional activation of genes encoding proteins required for adaptive response (Janget al. , Plant Mol. Biol. 37: 839-847, 1998; Liu et al., Science 280: 1943-1945, 1998; Pardo et al., Proc. Acad. Natl. Sci. USA 95: 9681-9686, 1998; Liu et al., Proc. Natl. Acad. Sci. USA 97: 3730-3734, 2000).

In order to understand the mechanism for adapting to osmotic stress, a number of genes whose expression is induced by specific stresses have been isolated and their characteristics studied extensively. As a result, various signaling systems exist that induce the expression of osmotic stress response genes (Jonak et al., Proc. Natl. Acad. Sci. USA 93: 11274-11279, 1996; Ishitani et al., Plant Cell 9 : 1935-1949, 1997), this signaling system comprises an ABA-dependent or ABA-independent pathway (La Rosa et al., Plant Physiol. 85: 174-181, 1987; Savoure et al., Mol. Gen. Genet. 254: 104-109, 1997), it has been found that certain signaling pathways work in common in all osmotic stress conditions such as cold, high salt and dehydration (Jang et al., Plant Mol. Biol. 37: 839-847, 1998; Liu et al., Science 280: 1943-1945, 1998; Pardo et al., Proc. Acad. Natl. Sci. USA 95: 9681-9686, 1998; Liu et al., Proc. Natl Acad.Sci. USA 97: 3730-3734, 2000).

LEA (Late Embryogenesis Abundant) protein has been known for more than 30 years, but its variety and its function are not exactly known (Wise and Tunnacliffe, 2004). LEA protein is highly expressed during plant seed development and accounts for about 4% of the maximum cellular protein. In addition, the expression is induced by low temperature, osmotic stress, ABA (abscisic acid) hormone. Under osmotic stress conditions, LEA protein function protects cellular structures by hydration, ion sequestration, direct protection of proteins or membranes, and protein regeneration (Bray, 1993).

The LEA protein is divided into six groups, of which the dehydrin protein belongs to group II and is present not only in plants but also in algae, yeast and cyanobacteria. Dihydrin proteins are characterized by being charged, high in polar amino acids, and having domains with repetitive sequences. The dihydrin protein has a conserved sequence of Lys-rich K-segment 'EKKGIMDKIKEKLPG' in the C-terminal region, which forms an amphipathic α-helix and stabilizes the protein in osmotic stress. Another well-conserved domain is the Y-segment in N-terminus, which has a conserved sequence of "DEYGNP" and is similar to the nucleic acid binding domain in plants and bacteria, but the exact function is not yet known. The last conserved domain is an S-segment consisting of eight Ser residues, which has been demonstrated to be phosphorylated in-flight.

LEA proteins expressed in yeast and enhanced osmotic stress resistance include EM protein belonging to wheat group I, LE25 protein belonging to tomato group III, and HVAI protein corresponding to group III of barley. The tomato LE4 protein belonging to group II showed similar growth to WT in the medium containing NaCl and sorbitorl when expressed in yeast, and the growth was better than the WT only in the medium containing KCl (Zhang et al. , J. Biochem. 127: 611-616, 2000). Osmotic stress resistance was enhanced by overexpression of wheat DHN-5 in Arabidopsis (Brini et al., 2007, Plant Cell Rep 26: 2017-2026), and the dihydrin genes RAB18 , COR47, LTI29 , LTI30 It was found that freezing resistance is enhanced in overexpressed transgenic plants expressing at once (Puhakainen et al., Plant Mol. Biol. 54: 743-753, 2004). The dehydrin gene PpDHNA of Physcomitrella patens , a moss, has been demonstrated to function as salinity and osmotic stress resistant (Saavedra et al., 2006, Plant J 45: 237-249).

However, until now, nothing was known about the dehydrin gene of Namunjae. The present inventors have identified the genes of SaDhn of Namunjae induced by various stresses and identified their function. When the SaDhn gene according to the present invention is overexpressed in yeast, the present invention was confirmed by the fact that NaCl (1 M), KCl (1 M), and sorbitol (1.5 M) -containing media have the function of enhancing osmotic stress resistance and freezing stress resistance. The invention has been completed.

It is an object of the present invention to provide a protein that enhances environmental stress resistance and a gene encoding the same.

Another object of the present invention to provide a recombinant vector comprising the gene.

Still another object of the present invention is to provide a composition for enhancing environmental stress resistance, comprising the gene as an active ingredient.

The present invention also provides a method of expressing the gene in plants to enhance resistance to environmental stress.

The present invention to solve the above problems is SaDhn derived from Namunjae Provide protein.

The present invention also provides a gene encoding the protein.

The present invention also provides a recombinant vector comprising the gene.

In another aspect, the present invention provides a composition for enhancing osmotic stress or freezing stress resistance comprising the gene.

In another aspect, the present invention provides a method for expressing the gene in plants to enhance resistance to osmotic stress or freezing stress.

According to the present invention, by overexpressing the SaDhn gene of the present invention can improve the resistance to osmotic stress and frozen stress, it is possible to improve the productivity of the plant.

1 is a diagram comparing protein sequences reported from other plants having homology with amino acid sequences deduced from cDNA sequences of SaDhn , a dehydrin gene of Namunjae . (The area marked with S is a domain consisting of Ser, and the line K is a domain conserved with 15 amino acids rich in Lys, consisting of 'EKKGIMDKIKEKLPG'.)
FIG. 2 is a graph comparing evolutionary distances by analyzing a protein sequence reported from another plant having homology with an amino acid sequence deduced from the cDNA sequence of SaDhn , a dehydrin gene of Namunjae , using the tree of life.
Figure 3 is a result of separating the total RNA from the high salt stress (0.2 M NaCl) -treated rape leaf and RT-PCR (A) and Northern blot analysis (B).
FIG. 4 is a diagram illustrating the expression of SaDhn , a dehydrin gene of Namuljae , in tobacco epidermal cells and protoplasts using GFP recombinant protein to analyze the intracellular location.
5 is yeast ( Saccaromyces) cerevisiae ) Expression vector pYES-DEST52 SaDhn cloned after the GAL1 promoter.
FIG. 6 compares growth for various osmotic stresses (1 M NaCl, 1 M KCl, 1.5 M sorbitol) by inoculating yeast with liquid medium SD (Ura-Raff 2%) or SD (Ura-Gal 2%). It is a graph.
FIG. 7 shows colonies growing resistant to various osmotic stresses (1 M NaCl, 1 M KCl, 1.5 M sorbitol) by inoculating yeast with solid medium SD (Ura-Raff 2%) or SD (Ura- Gal 2%). Compared through the photo.
8 is a graph comparing the survival rate by culturing yeast in solid medium SD (Ura-Glc 2%) before and after freezing stress.

In one embodiment, the present invention relates to SaDhn ( S uaeda ), a dehydrin gene belonging to the Late Embryogenesis Abundant (LEA) protein group II from Suaeda asparagoides . It provides a sparagoides D e h n ydri) protein.

The range of SaDhn protein according to the present invention includes a protein having an amino acid sequence represented by SEQ ID NO: 2 isolated from a palm tree and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2.

SaDhn proteins of the invention, as well as proteins having their native amino acid sequence, are also encompassed within the scope of the invention. Variants of SaDhn protein refer to proteins in which the SaDhn native amino acid sequence and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is known in the art (H. Neuroath, RL Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly. In some cases, it may be modified by phosphorylation, sulfation, acetylation, glycosylation, methylation, farnesylation, or the like.

The SaDhn protein or variant thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook et al. al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

Genes of the present invention include both genomic DNA and cDNA encoding SaDhn protein and genomic DNA and cDNA can be prepared according to methods known in the art.

Genomic DNA extracts genomic DNA from cells with, for example, the SaDhn gene and constructs a genomic library (vectors may be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and viewed the library. Colony or plaque hybridization is performed using a probe constructed on the basis of DNA encoding the protein of the invention (eg, SEQ ID NO: 1), or DNA encoding the protein of the present invention (eg, SEQ ID NO: 1). It can also be prepared by preparing a primer specific for the polymerase chain reaction (Polymerase Chain Reaction, PCR) using the same.

For example, cDNA synthesizes a cDNA based on mRNA extracted from a cell having a SaDhn gene, inserts the synthesized cDNA into a vector such as λZAP, prepares a cDNA library, and expands the cDNA library. Can be prepared by colony or plaque hybridization or by PCR.

Preferably, the SaDhn gene of the present invention may include a nucleotide sequence represented by SEQ ID NO: 1. In addition, variants of the above nucleotide sequences are included within the scope of the present invention. SaDhn nucleic acid molecules that can be used as the gene encoding the SaDhn protein of the present invention are functional equivalents of nucleic acid molecules constituting them, for example, Although some sequences of SaDhn nucleic acid molecules have been modified by deletion, substitution, or insertion, it is a concept that includes variants that can function functionally the same as SaDhn nucleic acid molecules. Specifically, the gene has a base sequence having a sequence homology of at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 1, respectively. It may include. "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

On the other hand, the gene encoding the SaDhn protein of the present invention may be provided included in a recombinant vector for intracellular delivery.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. Recombinant cells can also express genes found in natural cells, but the genes have been modified and reintroduced into cells by artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. The term "carrier" is often used interchangeably with "vector". The term “expression vector” refers to a recombinant DNA molecule comprising a coding sequence of interest and an appropriate nucleic acid sequence necessary to express a coding sequence operably linked in a particular host organism. The recombinant vector is preferably a recombinant yeast expression vector or a recombinant plant expression vector.

The expression vector preferably comprises one or more selectable markers. The marker is typically a nucleic acid sequence having properties that can be selected by chemical methods, and all genes that can distinguish transformed cells from non-transformed cells. Examples include herbicide resistance genes such as glyphosate or phosphinotricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol. However, the present invention is not limited thereto.

The present invention also provides a plant having enhanced osmotic stress or freezing stress resistance, transformed with a recombinant vector comprising the SaDhn gene of the present invention.

The plant according to the present invention is Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, Chinese cabbage, cabbage, gall radish, watermelon, It may be a dicotyledonous plant such as melon, cucumber, pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, pea, or monocotyledonous plant such as rice, barley, wheat, rye, corn, sugar cane, oats, and onions.

The present invention also provides a method of enhancing the osmotic stress or cryo stress resistance, comprising the step of transforming a plant with a recombinant vector comprising the SaDhn gene of the present invention to express the SaDhn gene. The plants are Arabidopsis, potato, eggplant, tobacco, pepper, tomato, burdock, garland chrysanthemum, lettuce, bellflower, spinach, chard, sweet potato, celery, carrot, buttercup, parsley, cabbage, cabbage, gatchi, watermelon, melon, cucumber, Dicotyledonous plants such as pumpkins, gourds, strawberries, soybeans, green beans, kidney beans and peas or monocotyledonous plants such as rice, barley, wheat, rye, corn, sugarcane, cane, oats and onions.

Transformation of a plant means any method of transferring DNA to a plant. Such transformation methods do not necessarily have a period of regeneration and / or tissue culture. Transformation of plant species is now common for plant species, including both terminal plants as well as dicotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Methods include calcium / polyethylene glycol methods for protoplasts (Krens, FA et al., Nature 296, 72-74, 1982), electroporation of protoplasts (Shillito RD et al., Bio / Technol. 3, 1099-1102, 1985), microscopic injection into plant elements (Crossway A. et al., Mol. Gen. Genet. 202, 179-185, 1986), particle bombardment methods (DNA or RNA-coated) of various plant elements (Klein TM) et al., Nature 327, 70, 1987), infection with (incomplete) virus in agrobacterium tumefaciens mediated gene transfer by plant infiltration or transformation of mature pollen or vesicles, and the like. Can be.

"Plant cell" used for transformation of a plant may be any plant cell. The plant cell may be any of a cultured cell, a cultured tissue, a culture or whole plant, preferably a cultured cell, a cultured tissue or culture medium, and more preferably a cultured cell.

The present invention also provides a composition for enhancing osmotic stress or freezing stress resistance comprising the SaDhn gene of the present invention.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example  One. SaDhn  ( S uaeda a sparagoides D e h ydri n )  The identification of

Suaeda asparagoides ) Seeds were distributed by Daejeon Biotechnology Research Institute. After germination, the leaves of Namunjae, grown in a greenhouse for more than 6 weeks, were soaked in NaCl (200 mM), treated for 0, 3, 6 and 24 hours, and then frozen in liquid nitrogen to extract total RNA (Chung et al., 2009 ). MRNA was isolated from total RNA, synthesized by cDNA, dsDNA synthesis, cloned into pBluescript-SK after adaptor linkage, and transformed into SOLR cells to prepare cDNA library (macrogen). A total of 932 items were analyzed in Namunjae EST library. After sequencing the obtained gene, gene information was collected using NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). By sequencing, the entire nucleotide sequence of the SaDhn gene was obtained.

As a result of sequencing, the cDNA of the dehydrin gene was composed of a total of 741 bp sequences (SEQ ID NO: 1), and deduced 246 amino acids (SEQ ID NO: 2). The base sequence and deduced amino acid sequence was found to be a new gene by database search.

Similarity analysis of the amino acid sequence encoded by the SaDhn gene by database search showed that the protein sequence most similar to the protein encoded by the gene was Arabidopsis. thaliana ) gene (GenBank ID: NP_177745) has a very low homology of 53%.

Through the amino acid sequence analysis obtained from the gene, it was found that there are S-segments having multiple Sers and three well-preserved K-segments called 'EKKGIMDKIKEKLPG' (FIG. 1). In addition, by analyzing the protein which is determined to have high homology with the amino acid sequence encoded by the SaDhn gene as a tree of life, the evolutionary relationship was examined (Fig. 2). The SaDhn -encoded protein and Arabidopsis dehydrin were closest to each other, and the homology with the dihydrin protein found in other plant types was very low (36-47%). Is thought to be very far from evolutionary. The genes used for protein comparison and tree analysis are as follows.

Rhododendron catawbiense (Gene bank ID: NP_177745); Coffea canephora (ABC68275); Cichorium intybus (ACF15448); Solanum chilense (ADQ73953); S. peruvianum (ADQ74000); Pisum sativum (CAA78515); Phaseolus vulgaris (AAB00554); Populus maximowiczii (ABS12346); Lupinus albus (AAT06600); Panax ginseng (ABF48477); Daucus carota (Q9XJ56)

Example  2. New Dihydrin  ( dehydrin )  gene, SaDhn Expression test according to salinity stress treatment of

Whole RNA was sampled using a high salinity (0.2 M NaCl) stress-treated leaf over time for six weeks or longer, and RNA was separated using RNA extraction kit (Ambion).

Thereafter, cDNA was synthesized with Total RNA (2 μg), and RT-PCR was performed using cDNA prepared for each sample as a template. The housekeeing gene, tubulin expression, was the front 5'tubulin primer (5'-ATGAGGGAGTGCATTTCAATCCA-3 '), the rear 3'tubulin primer (5'-AAGAACACTGTTGTAGGGCTCC-3'), and the front 5'SaDhn to analyze SaDhn expression. As primer (5'-ATGGCAGATGAAAGGATTAGTGA-3 '), the rear 3'SaDhn primer (5'-GTGCACCTCCTCATGAGTGTGG-3') was used, and PCR conditions were 5 minutes (denaturation) at 95 ° C; 1 minute (denaturation) at 95 ° C., 1 minute at 55 ° C. (annealing, 1 minute at 72 ° C.) was performed under conditions of 26 cycles; 7 minutes (final extension) at 72 ° C. (FIG. 3A ).

Northern blot analysis was performed as follows. The total RNA isolated was electrophoresed on a 1.0% formaldehyde gel, and then the gel was transferred to a nylon membrane (purchased by AP Biotech) with 20 × SSC solution (3 M NaCl, 0.3 M Sodium citrate) and radioisotope. Analysis was performed using a fragment 741 bp cDNA of the dehydrin gene labeled with the element [α-32P] dCTP as a probe (FIG. 3B).

As a result, it was confirmed that SaDhn transcripts gradually increased due to salt stress.

Example  3. Plant Expression Vector  using GFP  Recombinant Fusion Protein Expression

In order to determine the intracellular location of the GFP fusion protein expressed in plants, GFP was used in the plant expression binary vector.

Using the entire SaDhn gene cDNA isolated in Example 1 as a template, PCR was performed using CACC-SaDhn primer (5'-CACCATGGCAGATGAAAGGATTAG-3 ') and 3NS-SaDhn primer (5'-ATATCCTTCCTTTTTCTCCTCATGGT-3') and amplified PCR. The product (742 bp) was cloned into the pENTR vector (purchased by Invitrogen) and directional PCR was cloned to confirm the sequence. For the base sequence confirmation pENTR-SaDhn plasmid with GFP targeted gateway vector of pK7FWG2 (Plant Systems Biology Inc. purchased) plasmid and LR Recombinase (Invitrogen Corporation purchased) and reacted SaDhn coding region The GFP of the N-terminal portion and the in-frame Fused. SaDhn-GFP fusion protein expression was confirmed in tobacco plants. The analysis of SaDhn protein of Namunjae using TargetP (http://www.cbs.dtu.dk/services/TargetP/), a protein location analysis program, predicted that SaDhn protein is mainly targeted to the nucleus (0.7). The intracellular location of SaDhn-GFP fusion protein was expressed in tobacco epidermal cells and protoplasts. As a result, it was found that the SaDhn-GFP fusion protein was expressed around the nucleus and cytoplasm (FIG. 4).

Example  4. SaDhn  Construction of yeast expression vector of gene

PYES-DEST52 (purchased by Invitrogen), a yeast expression vector, was used to express in yeast (FIG. 5). SaDhn isolated in Example 1 PYES-Amplified PCR product (742 bp) by PCR amplification with CACC-SaDhn primer (5'-CACCATGGCAGATGAAAGGATTAG-3 ') and 3NS-SaDhn primer (5'-ATATCCTTCCTTTTTCTCCTCATGGT-3') using the entire gene cDNA as a template. After directional PCR cloning into DEST52 vector (Invitrogen) purchased, the sequence was confirmed. In the case of pYES-GFP, CACC-5GFP primer (5'-CACCATGAGTAAAGGAGAAGAACT-3 ') and 3-GFP primer (5'-TTATTTGTATAGTTCATCCATGTG-3) were used as a template for the pYES-GFP. PCR products amplified with ') (700 bp) was directional PCR cloned into the pENTR vector (Invitrogen) and confirmed the base sequence. The nucleotides pENTR-SaDhn and pENTR-GFP, respectively, identified as nucleotide sequences, were reacted with pYES-DEST52 (purchased by Invitrogen), a plasmid and yeast expression vector, and LR Recombinase (purchased by Invitrogen), followed by SaDhn and GFP coding regions. Cloning and DNA sequencing confirmed the sequence. pYES-GFP and pYES-SaDhn by transforming each of the plasmids in EGY48 cell with LiAc method SD (Ura-deficient) were cultured for 2 days in 30 ℃ then plated on selective medium, the colonies obtained by the culture results as template, SaDhn Transformation was confirmed by colony PCR using gene primers.

Example  5. SaDhn  Osmotic stress resistance assay using yeast expression vector of gene

Normal WT yeast (pYES-GFP) and SaDhn- expressing yeast (pYES-SaDhn) were incubated overnight in SD (Ura-, Raff 2%) liquid medium and fresh SD (Ura-, Raff 2%) or SD (Ura-). , Gal 2%) suspended in a liquid medium to measure the OD was adjusted to 0.2, OD was measured while incubating at 28 ℃ (Fig. 6).

In order to analyze the osmotic stress resistance, NaCl (1 M), KCl (1 M), and sorbitol (1.5 M) were added to the liquid medium to inoculate WT yeast and SaDhn- expressing yeast in the same manner as above. Were measured to compare growth (Fig. 6B-D).

In addition, normal WT yeast (pYES-GFP) and yeast (pYES-SaDhn) expressing SaDhn in solid medium were incubated in SD (Ura-, Raff 2%) liquid medium overnight after incubation. Ura-, Raff 2%) After measuring the OD in the liquid medium and adjusting it to 0.7, dilute it with 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 with sterile distilled water and dispense 10μl into the solid medium. Cancer culture was carried out for 3 days at 30 ℃ (Fig. 7).

As a result, the yeast (pYES-SaDhn) strains expressing SaDhn than WT (pYEST-GFP) yeast was confirmed that a much better growth in osmotic stress.

Example  6. SaDhn  Frozen Stress Resistance Assay Using Yeast Expression Vectors of Genes

Normal WT yeast (pYES-GFP) and SaDhn expressing yeast (pYES-SaDhn) were incubated overnight in SD (Ura-, Raff 2%) liquid medium and suspended in new SD (Ura-, Raff 2%) liquid medium. Measure OD to 0.7, dilute to 10 ^-4 with sterile distilled water, dispense 50μl in SD (Ura-, Glc 2%) solid medium, smear and incubate for 3 days at 30 ℃ Counted. In addition, WT (pYES-GFP) yeast and SaDhn- expressing yeast (pYES-SaDhn) were cultured in SD (Ura-, Raff 2%) liquid medium overnight to investigate their growth after freezing stress. , Raff 2%), suspended in liquid medium, OD is measured to 0.7, and then the Eppendorf tube containing yeast is immersed in ethanol (100%) and frozen in -20 ° C freezer for 24 hours and then 30 ° After thawing in C waterbath for 10 minutes, frozen again at -20 ° C freezer for 24 hours, then thawing in 30 ° C waterbath for 10 minutes, diluted with distilled water to 10 ^-4 and then SD (Ura-, Glc 2%) solid medium After dispensing 200μl each, smeared and cultured at 30 ° C for 3 days, the number of colonies was counted. Survival rate was calculated as the percentage of the number of colony after freezing stress to the number of colony before freezing stress, and the average value of three repeated experiments was calculated (FIG. 8).

As a result, it was confirmed that the yeast (pYES-SaDhn) strain expressing SaDhn than the WT (pYEST-GFP) yeast had a survival rate about 2 times higher even with frozen stress.

<110> Dong-A University Research Foundation For Industry-Academy Cooperation <120> Genes enhancing resistance to stress and uses <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 741 <212> DNA <213> Suaeda asparagoides <400> 1 atggcagatg aaaggattag tgaggcaaca cccggagttg tcgaaaccac tgaccgtggt 60 ttatttgatt tcatgaagaa gaaggaagat gagtctaagc cggcttcatc ggagtatgat 120 aatatcaact ccggtgtcga aaaggtacac atatcggaac ccgagtataa ggaagaacat 180 catcattatg aggagaagaa gcatgaaagt cttggtgaaa agcttcatcg ctctaatagc 240 tcaagctcat ctagctcctc tgatgaagaa ggagatgatg aggagaagaa gaggagaaga 300 aaggaaagaa aagagaagaa gaaaggagga ataaaggaaa aaattgagga aaaatttggt 360 catcatgatc acaaagaaga aaaacatcat gaacatgata caaatgtacc aattgagaaa 420 attcatgtgc aagagaatgt ttattctgag ccatcataca ctacacatgc acatgatcat 480 catgaggagg agaaaaagaa gggtggtttc atggacaaga ttaaggacaa gttgccaggt 540 cagcacggcg acgaggagca caaggtggct gcaccggtgg tgagccacac tcatgaggag 600 gtgcacggcg ccgaggagga gaaggagaag aagggattct tggacaagat taaggagaag 660 atacctggat tccactctaa gaatggtgct gaggagaaga aagagcatga tcaccatgag 720 gagaaaaagg aaggatatta a 741 <210> 2 <211> 246 <212> PRT <213> Suaeda asparagoides <400> 2 Met Ala Asp Glu Arg Ile Ser Glu Ala Thr Pro Gly Val Val Glu Thr   1 5 10 15 Thr Asp Arg Gly Leu Phe Asp Phe Met Lys Lys Lys Glu Asp Glu Ser              20 25 30 Lys Pro Ala Ser Ser Glu Tyr Asp Asn Ile Asn Ser Gly Val Glu Lys          35 40 45 Val His Ile Ser Glu Pro Glu Tyr Lys Glu Glu His His His Tyr Glu      50 55 60 Glu Lys Lys His Glu Ser Leu Gly Glu Lys Leu His Arg Ser Asn Ser  65 70 75 80 Ser Ser Ser Ser Ser Ser Ser Asp Glu Glu Gly Asp Asp Glu Glu Lys                  85 90 95 Lys Arg Arg Arg Lys Glu Arg Lys Glu Lys Lys Lys Gly Gly Ile Lys             100 105 110 Glu Lys Ile Glu Glu Lys Phe Gly His His Asp His Lys Glu Glu Lys         115 120 125 His His Glu His Asp Thr Asn Val Pro Ile Glu Lys Ile His Val Gln     130 135 140 Glu Asn Val Tyr Ser Glu Pro Ser Tyr Thr Thr His Ala His Asp His 145 150 155 160 His Glu Glu Glu Lys Lys Lys Gly Gly Phe Met Asp Lys Ile Lys Asp                 165 170 175 Lys Leu Pro Gly Gln His Gly Asp Glu Glu His Lys Val Ala Ala Pro             180 185 190 Val Val Ser His Thr His Glu Glu Val His Gly Ala Glu Glu Glu Lys         195 200 205 Glu Lys Lys Gly Phe Leu Asp Lys Ile Lys Glu Lys Ile Pro Gly Phe     210 215 220 His Ser Lys Asn Gly Ala Glu Glu Lys Lys Glu His Asp His His Glu 225 230 235 240 Glu Lys Lys Glu Gly Tyr                 245

Claims (7)

SEQ ID NO: consisting of the amino acid sequence of Figure 2, gave the di-derived namunjae high SaDhn (S uaeda a sparagoides D e h n ydri) protein. The gene encoding the SaDhn protein of claim 1. A recombinant vector comprising the gene of claim 2. Composition for enhancing resistance to osmotic stress of plants comprising the gene of claim 2. Composition for enhancing resistance to freezing stress of plants comprising the gene of claim 2. A method of enhancing resistance to osmotic stress in a plant, comprising the step of transforming the plant with the recombinant vector of claim 3 to express the SaDhn gene. A method of enhancing resistance to freezing stress in a plant, comprising the step of transforming the plant with the recombinant vector of claim 3 to express the SaDhn gene.

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